PROJECT SUMMARY Several landmark discoveries in cancer genetics have come from studies on a rare childhood cancer of the developing retina called retinoblastoma. In parallel, advances in preclinical testing and clinical research has led to improvements in outcome for children with this devastating disease. Despite these advances, there are still fundamental questions in the retinoblastoma field that remain unanswered. Are retinal cells fully transformed once they sustain biallelic inactivation of the RB1 gene or is retinoblastoma tumorigenesis a multistage process? Why do some family members with the same germline RB1 mutation have bilateral multifocal retinoblastoma at a young age while others have no evidence of disease? Can treatment induce a process of tumor cell clonal evolution and selection that leads to tumor progression and enucleation? These questions have been impossible to answer because retinoblastomas are not biopsied and enucleation is only performed for advanced stage eyes. In order to overcome this barrier in the field, we have developed the first spontaneous human retinoblastoma tumor model using 3D retinal organoids produced from patients with germline RB1 mutations. I have assembled a multidisciplinary team with expertise in computational and stem cell biology, oncology, pathology, epidemiology and biostatistics to use this innovative new model of retinoblastoma to answer fundamental questions in 3 specific aims. We will determine if retinoblastoma progresses through a multistep process (Aim 1), if molecular, cellular or genetic factors contributes to differences in penetrance and expressivity (Aim 2) and if there is clonal selection with treatment (Aim 3). I have a proven record in retinoblastoma genetics and of moving basic science discoveries into clinical trials. This proposal will impact patients with retinoblastoma through preclinical testing of a novel maintenance therapy (Aim 3) to prevent new tumors from forming in the peripheral retina in the first few months after completion of chemotherapy. It may also help to identify a subset of retinoblastoma survivors with germline RB1 mutations that have an ultra-high risk of developing a 2nd malignancy and require more extensive cancer screening (Aim 2). No other center has the team, resources, expertise, or tools available to perform the studies presented here and efficiently move the most promising findings directly into a clinical trial.